RNAPack: An Integrated NMR Approach to RNA Structure Determination

Abstract

Over the last decade, a vast number of useful nuclear magnetic resonance (NMR) experiments have been developed and successfully employed to determine the structure and dynamics of RNA oligonucleotides. Despite this progress, high-resolution RNA structure determination by NMR spectroscopy still remains a lengthy process and requires programming and extensive calibrations to perform NMR experiments successfully. To accelerate RNA structure determination by NMR spectroscopy, we have designed and programmed a package of RNA NMR experiments, called RNAPack. The user-friendly package contains a set of semiautomated single, double, and triple resonance NMR experiments, which are fully optimized for high-resolution RNA solution structure determination on Varian NMR spectrometers. RNAPack provides an autocalibration feature that allows rapid calibration of all NMR experiments in a single step and thereby speeds up the NMR data collection and eliminates user errors. In our laboratory, we have successfully employed this technology to solve RNA solution structures of domains of the internal ribosome entry site of the genomic hepatitis C viral RNA in less than 3 months. RNAPack therefore makes NMR spectroscopy an attractive and rapid structural tool and allows integration of atomic resolution structural information into biochemical studies of large RNA systems.